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Nucleic Acids 5Control of Gene Expression and Recombinant DNA Technology (Cloning)

Objectives & Handout

Objectives:

I. Control of Gene ExpressionA. Control of Gene Expression General Comments.1. Describe Constitutive or Housekeeping Genes.2. Describe Regulated or Inducible Genes.

a) Describe Regulator Sequence.b) Describe Repressor.c) Describe Inducer.

3. Describe the four general mechanism for controlling the expression of genes.B. The E. coli Lac Operon; An Example of Control of Transcription in Prokaryotes.

1. De ne the term operon.2. Describe the structure of the Lac operon.

3. Describe the function of the proteins coded for by this operon.4. Describe how the Lac operon function in the presence of glucose and absence of galactose5. Describe how the Lac operon function in the absence of glucose and presence of galactose6. Describe how the Lac operon function in the presence of both glucose and galactose

a) Describe Catabolite Activationb) What is cAMP Receptor Protein (CRP)?c) What is Catabolite Activator Protein (CAP)?d) What are their functions?

C. Control of Transcription in EukaryotesD. Describe the control of transcription at the level of chromatin structure.

1. What is Heterochromatin?

2. What is Euchromatin?E. Describe the control of transcription at Initiation of Transcription.

II. Recombinant DNA: Cloning and Creation of Chimeric GenesA. Describe the Polymerase Chain Reaction.

1. How is it useful?2. What are its limitations?3. Why is Taq Polymerase more useful than the other DNA polymerases?

B. Describe Restriction Fragment Length Polymorphisms (RFLP).1. What are Hypervariable Regions?2. How are Hypervariable Regions useful for RFLP?

C. Describe the overall strategy of genetic engineering for introducing a gene into an organism.1. Library Synthesis

a) Describe the synthesis of Genomic DNA Library.b) Describe the synthesis of a cDNA Library.

(1) What are the differences between the two types of libraries?(2) What are the different possible uses of the two different types of libraries?

2. Fragment Generation Sticky Ends.a) Describe a Restriction Endonuclease.b) What is its normal function within a bacterial cell?

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c) What function do Molecular Biologists use these enzymes for?d) What is a sticky end?e) Describe why Genomic Libraries are treated with Restriction Endonucleasesf) Describe why the ends of DNA fragments obtained from cDNA libraries are modi ed.

3. Joining of DNA to a Cloning Vehicle - Forming the Recombinant Vector.a) Describe the properties of Vectors.

(1) What four DNA sequences are important for a good vector?(2) What are the differences between a phage vector, plasmid vector, and a lab

constructed vector.b) Describe the general process of opening a vector and ligating the foreign DNA to the

vector.4. Insertion into Host Cell.

a) Describe the general process for introducing the recombinant vector into a compatiblehost cell.(1) What is Transformation?(2) What is Transfection?

5. Identifying cells that have taken up the recombinant vector.

a) Describe how cells that have taken up the recombinant vector are selected for.(1) Why is a double marker better than a single marker?6. Identifying cells that contain the recombinant vector of interest.

a) Describe how cells that contain the recombinant vector of interest are selected for.7. Replication &/or expression of the recombinant DNA

a) Now that you have a recombinant cell what can be done with it.b) Expression in eukaryotic cells.

(1) Why must some protein be expressed in eukaryotic cells rather than bacteria?(2) What is a Shuttle Vector?(3) What DNA sequences are important for a good shuttle vector?

8. Recognize the potential of pharmaceuticals produced by recombinant DNA.



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Promoter Gene

RNA Polymerase

Activator

Signal Molecule

Signal Molecule



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Promoter Gene

Repressor Protein

Signal Molecule

RNA Polymerase Repressor Protein Signal Molecule

Complex

Repressor Protein

Signal Molecule

RNA Polymerase



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Lac OperonP I P Lac

Lac I O 1 O 2

Lac Z Lac Y Lac A

ter

Lac I O 1 O 2 Lac Z Lac Y Lac A P I P L

Lac I O 1 O 2 Lac Z Lac Y Lac A P I P L

Lac I O 1 O 2 Lac Z Lac Y Lac A P I P LcAMPcAMP

Lac I O 1 O 2 Lac Z Lac Y Lac A P I P LcAMPcAMP



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AAAAAAAAP-5 3

AAAAAAAAP-5 3

TTTTTTTTHO-

Oligo dT PrimerDeoxynucleoside TriphosphatesReverse Transcriptase

TTTTTTTTHO-

RNase H

P-5 3

HO-

DNA Polymerase I

P-5 3

HO-

T4 DNA Polymerase

-OH-P

-OH-P

P-5 3

HO-

Oligonucleotide LinkersDNA Ligase

-OH-P

5 3

HO-

Resctiction Endonuclease

-OHP--P



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1. DNA strands separated by heating.2. Primers bind (anneal) to complementary sequences.

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3. Taq DNA Polymerase extends (synthesizes)complementary DNA

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53 5

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5 3

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5 3

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1. DNA strands separated by heating.2. Primers anneal to complementary sequences.3. Taq DNA Polymerase synthesizes DNA.

Cycle 2

1. DNA strands separated by heating.2. Primers anneal to complementary sequences.

3. Taq DNA Polymerase synthesizes DNA.

Cycle 3



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Step 1 - The gene of interestis identified and an assayfor its detection developed.Total mRNA is isolated fromhuman cells and the mRNAcorresponding to the geneof interest isolated.

Step 2 - mRNA is used as a templateto synthesize double stranded DNAusing Reverse Transcriptase.

Step 3 - Sequences specificfor one of the RestrictionEndonucleases are added tothe DNA fragments usingDNA Ligase .

Step 4 - An appropriatevector is chosen.

Step 5 - The vector and DNAfragment are cut with theappropriate Restriction

Endonuclease to generatesticky ends. By complimentarybase pairing the DNA fragment(recombinant gene) interacts withthe opened vector.



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Step 6 - Vector and Recombinant

DNA fragment treated with DNALigase to seal the pieces together.The human gene is now part of therecombinant vector.

Step 7 - The cells are transfectedwith the recombinant vector. Thevector is replicated and passed toeach daughter cell during cell division.

Step 8 - The cells containing therecombinant vector aredetected and identified,usually by resistance to someantibiotic. Cells containingthe recombinant vectorare grown in pure culture.



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Transcription

Translation

Secretion



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Step 1 - DNA containing samples are collected from the crime scene. These samples willcontain DNA from the victim, from the criminal, and from extraneous sources.

Step 2 - Cells are collected from the victim and possible suspects. A small blood sample issufficient.

Step 3 - The DNA is isolated from all of the samples.

Step 4 - The DNA is digested with one of the Restriction Endonucleases . Thsi digestioncleaves the DNA into fragments of random size.

Step 5 - DNA fragments are separated according to size by electrophoresis on an agarosegel matrix.

Step 6 - The gel is incubated with the probe DNA . This is a single stranded piece of DNAcontaining the sequence from one of the hypervariable regions of the human genome.

Step 7 - The fragment pattern from the crime scene samples are compared to the fragment pattern of the victim and the suspects.

Step 8 - Matching patterns indicate identity.

Step 9 - The experiment may be repeated using a different Restriction Endonuclease and/or a different probe, a probe specific for a different hypervariable region of thegenome.

MWM Con V E1 E2 S1 S2


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